Project Summary/Abstract The failure of cancer therapies to achieve durable responses is often attributed to intratumoral heterogeneity (ITH), which fosters tumor evolution and the generation of therapy-resistant clones. ITH has historically been assessed by genomic alterations such as somatic mutation and copy-number alteration. Recent studies suggest that epigenomic ITH may also contribute to tumor evolution and therapy resistance. However, the prevalence and degree of epigenomic ITH are not well understood. Low-grade gliomas (LGGs), which include grade II astrocytoma and grade II oligodendroglioma, are slow-growing tumors treated with surgical resection, and in some cases, with radiation and temozolomide chemotherapy. These tumors inevitably recur as grade III astrocytoma and grade III oligodendroglioma, or as grade IV secondary glioblastoma. LGGs are characterized by clonal mutations in isocitrate dehydrogenase (IDH) genes, which drive production of the oncometabolite D- 2-hydroxyglutarate (2HG) and result in epigenomic alterations including the development of the glioma CpG island methylator phenotype (G-CIMP). Data published by our laboratory and preliminary data presented here indicate that the epigenomes of IDH-mutant gliomas display ITH across spatially distinct tumor regions and show evolution over time. In this proposal, we will test the central hypothesis that epigenomic ITH is a feature of IDH-mutant gliomas that is biologically and clinically significant. For a cohort of IDH-mutant glioma patients, we will characterize ITH in three-dimensional space using a novel topographic approach developed in collaboration with neurosurgeons, neuropathologists, and biomedical imaging experts. We will profile DNA methylation and chromatin accessibility, two complementary approaches towards characterizing the epigenomic state of the cell. In Aim 1, we will investigate the spatial patterning of epigenomic ITH relative to genomic ITH. In Aim 2, we will determine the biological significance of epigenomic ITH by investigating its relationship with gene regulation. In Aim 3, we will determine the clinical significance of epigenomic ITH by investigating its relationship with tumor histologic features and immune cell content, and by investigating ITH of DNA-methylation based biomarkers including MGMT methylation and epigenetic age. Collectively, these studies will provide the most comprehensive characterization of epigenomic ITH to date. Knowledge gained from these studies will deepen our understanding of the contribution of epigenomic ITH to tumor evolution and therapy resistance, ultimately guiding the design of novel and improved approaches towards countering therapy failure. The results of this study may also directly impact clinical practice in the treatment of IDH- mutant glioma by supporting the use of epigenomic signatures in glioma grading and diagnostics and by guiding the use of immunotherapeutics and biomarkers for patient stratification.